Xu et al. Bot Stud (2017) 58:56 DOI 10.1186/s40529-017-0207-5 ORIGINAL ARTICLE Open Access Three new species of Paraboea (Gesneriaceae) from limestone karsts of China based on morphological and molecular evidence Wei‑Bin Xu1, Jing Guo2, Bo Pan1, Meng‑Qi Han3, Yan Liu1* and Kuo‑Fang Chung4* Abstract Background: The limestone karsts of Southeast Asia and South China are a major biodiversity hotspot of global terrestrial biomes. With more than 130 described species, Paraboea has become one of the most characteristic plant groups in the Southeast Asian limestone fora. During the course of extensive feld work on the limestone formations of southern and southwestern China, three unknown species of Paraboea were collected. Results: Molecular phylogenetic analyses based on nuclear ITS and chloroplast trnL-F sequences strongly con‑ frm the placements of the three new species in Paraboea sensu Puglisi et al. (Taxon 65:277–292. https://doi. org/10.12705/652.5, 2016). Moreover, these three novelties can be distinguished from known Paraboea species with distinct morphological characters, further supporting their recognition as new species. Conclusions: With the support of detailed morphological studies and molecular phylogenetic analyses, Paraboea dushanensis, P. sinovietnamica and P. xiangguiensis are recognized as three species new to science. Keywords: Limestone fora, Loxocarpinae, Paraboea dushanensis, Paraboea sinovietnamica, Paraboea xiangguiensis, Sino-Vietnamese limestone karsts (SVLK) Background Malesia as far east as Sulawesi (Middleton et al. 2010). As currently circumscribed, the Asian gesneriad genus Since the last major revision by Xu et al. (2008) in which Paraboea (C.B.Clarke) Ridl. comprises ca. 130 species of 89 species and 5 varieties were recognized, Paraboea has rosulate or caulescent herbs characterized by the abaxi- been expanded to include the ca. 20 species of Phylloboea ally matted leaves with densely interwoven indumentum Benth. and Trisepalum C.B.Clarke (Puglisi et al. 2011), and fowers with fat-faced to shortly campanulate corolla with the reduction of fve taxa constituting the new genus and non-erect anthers (Middleton et al. 2010; Puglisi et al. Middletonia C.Puglisi (Puglisi et al. 2016). More than 2016). A majority of Paraboea species are lithophytes on 30 new species have also been described since the revi- limestone substrates, distributed in South China, north- sion by Xu et al. (2008), almost all narrowly distributed eastern India and the eastern Himalayas, Indochina, and endemic from limestone karsts (Chen et al. 2008, 2012; Kiew 2010; Triboun and Middleton 2012, 2015; Xu et al. 2012a; Triboun 2013; Wen et al. 2013; Puglisi et al. 2015; *Correspondence: [email protected]; [email protected] Guo et al. 2016; Wen and Wei 2016). Because a great pro- 1 Guangxi Key Laboratory of Plant Conservation and Restoration portion of Asian limestone karsts remain unexplored or Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuangzu Autonomous Region and Chinese Academy of Sciences, Guilin 541006, underexplored, it is fully expected that additional new China species of Paraboea will be unearthed given that further 4 Research Museum and Herbarium (HAST), Biodiversity Research Center, feld investigations and herbarium work are conducted Academia Sinica, Taipei 11529, Taiwan Full list of author information is available at the end of the article (Puglisi et al. 2015). © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Xu et al. Bot Stud (2017) 58:56 Page 2 of 14 Sino-Vietnamese limestone karsts (SVLK) are vast ter- and directly sequenced. Sequencing reactions were per- rains striding across the border between China and Viet- formed using the ABI Prism Bigdye Terminator Cycle nam (Xu et al. 2012b; Chung et al. 2014), renowned for Sequencing Kit (Applied Biosystems, Foster City, Califor- their spectacular landscape and rich biodiversity (Myers nia, USA). Sequences were analyzed using an ABI 3730 et al. 2000; Clements et al. 2006; Hou et al. 2010; López- DNA Sequencer. Te program Sequencher 5.0 (Gene Pujol et al. 2011). Botanically, the SVLK are home to a Codes Co., Ann Arbor, Michigan, USA) was used to eval- myriad of species-rich genera with narrowly endemic uate chromatograms for base confrmation and to edit entities (Chung et al. 2014) such as Aspidistra Ker- contiguous sequences. Sequences were initially aligned Gawler (e.g., Liu et al. 2011, 2016), Begonia L. (e.g., Peng using MUSCLE 3.8.31 (Edgar 2004), followed by manual et al. 2014, 2015), Elatostema Forster & Forster (e.g., Wei adjustments in Geneious 9.1.2 (http://www.geneious. et al. 2011), Impatiens L. (e.g., Tan et al. 2015; Yu et al. com, Kearse et al. 2012). 2015), Polystichum Roth (He and Zhang 2011; Zhang and He 2011), and several genera of Gesneriaceae (Wei 2010; Phylogenetic analyses Xu et al. 2012b, 2014; Guo et al. 2015) including Para- Te phylogenetic analyses were conducted based on boea (Xu et al. 2012a; Guo et al. 2016). maximum likelihood (ML) and Bayesian inference (BI) During the course of extensive foristic surveys in methods for the individual locus datasets (ITS/trnL-F) limestone karsts of southern and southwestern China in and combined dataset (ITS-trnL-F), using RAxML v7.0.4 recent years, we collected three species of Paraboea with (Stamatakis et al. 2008) and MrBayes v3.3.5 (Ronquist spectacular fowers and/or fruits not known previously. et al. 2012), respectively. Te model GTR + Ґ was selected After consulting the relevant literature (Burtt 1984; Wang as the optimal model for both DNA regions based on et al. 1990, 1998; Li and Wang 2004; Chen et al. 2008, the Akaike Information Criterion via jModeltest v2.1.4 2012; Xu et al. 2008, 2012a; Kiew 2010; Triboun and Mid- (Posada 2008). For ML analyses, node support was esti- dleton 2012; Wen et al. 2013; Guo et al. 2016; Wen and mated with nonparametric bootstrap (1000 replicates) Wei 2016), as well as herbarium specimens of E, GXMI, following a thorough search for the best ML tree. For BI HITBC, IBK, IBSC, KUN, and PE (herbarium acronyms analyses, four runs of Metropolis-coupled Markov chain according to Index Herbariorum; Tiers 2017), they were Monte Carlo (MCMCMC) analyses were conducted with identifed as three new species of Paraboea based on one tree sampled for every 2000 generations over 20 mil- detailed examination of salient morphological and ana- lion generations, starting with a random tree. Analyses tomical features and molecular phylogenetic analyses. were run until the average standard deviation of the split frequencies approached 0.01, indicating that two runs Methods converged to a stationary distribution. Te frst 25% of Taxon sampling and DNA sequencing sampled trees corresponding to the burn-in period was For phylogenetic analyses, a majority of species of Para- discarded, and the remaining trees were used to construct boea available in GenBank were used, with nine addi- a majority-rule consensus tree. We used bootstrap sup- tional species endemic to China sampled. A total of 83 port (BS) ≥ 70% and posterior probability (PP) ≥ 0.95 as accessions representing 67 species of Paraboea were the thresholds for strongly supported clades (Wang et al. included in this study. Based on Puglisi et al. (2016), 2014). To investigate congruence between the nuclear two species of Ornithoboea Parish ex C.B.Clarke and and chloroplast genomes, topologies of the ITS and trnL- three species of Middletonia C.Puglisi were chosen as F datasets of both ML and BI analyses were compared. outgroups. Species, voucher information, and NCBI Because a majority of clades with BS ≥ 70% and PP ≥ 0.95 accession numbers are listed in Additional fle 1. Two were congruent without signifcant conficts, the concat- molecular markers, including the nuclear ITS (internal enated dataset was presented for further discussion. transcribed spacer) and the chloroplast trnL-F intron- spacer region (trnL-F), were used in this study. Total Results and discussion genomic DNA was extracted from silica gel-dried leaf Te concatenated DNA matrix (78 ITS sequences and materials using the CTAB protocol (Doyle and Doyle 81 trnL-F sequences) had a length of 1780 aligned char- 1987). Te primers ITS-4 and ITS-5 (Möller and Cronk acters (ITS: 812 bp, trnL-F: 968 bp), of which 645 (ITS: 1997) were used to amplify and sequence the ITS region. 454 bp, trnL-F: 191 bp) are variable and 415 (ITS: 332 bp, Te primers trnL-F e and f (Taberlet et al. 1991) were trnL-F: 83 bp) are parsimony informative. Te best ML used to amplify and sequence the cp DNA region based phylogram with bootstrap (BS) supports and posterior on the PCR procedures outlined in Guo et al. (2016). probability (PP) values of Bayesian analyses is depicted in Te PCR products were purifed using the Tian quick Fig. 1. Te phylogenetic relationships of the concatenated Midi Purifcation Kit (TianGen Biotech, Beijing, China) matrix are congruent with those reported in Puglisi et al. Xu et al. Bot Stud (2017) 58:56 Page 3 of 14 (2011, 2016). Samples of the three new species (Para- ferrugineous matted indumentum outside and glabrous boea dushanensis, P. sinovietnamica and P. xiangguiensis) inside; pedicels 0.6–1.5 cm long, ca. 0.7 mm in diameter, are shown as distinct clades grouped within Paraboea covered with ferrugineous matted indumentum. Calyx sensu Puglisi et al. (2016) with strongest support values 3.5–5 mm long, 5-parted nearly to the base, lobes linear- (BS = 100%, PP = 1.00), ascertaining their recognition lanceolate, 1.0–1.2 mm wide, with ferrugineous mat- as distinct species of Paraboea.